DNA smears after digestion - DNA smears after digestion (Nov/26/2007 )
Hi;
I hope somebody can help me with what I've been going thruogh for the last 5 weeks. I have been getting smears of DNA after digesting my plasmid with different enzymes. I even dont have to put an enzyme and jsut by adding a bfufer I get smears. I did this for 2 hours and O/N incubation and still the same thing. I tried using a plasmid that I knew it worked ebfore and I got smears again for reasons I dont know. I boiled the plasmid to make sure to get rid of DNA it didnt help. I changed all water, buffer, tubes, tips, bench utensils and still I am ahving this problem. I re-transformed and re-purified the DNA (using QIAGEN miniprep kit) yet I am still havingt the same problem. The uncut looks perfect when I just add water (no buffer or BSA) however as soon as I add buffer and incubate it, I get smears. It is jsut crazy. It is something truly unbelievable. I have literally ran more tahn 200 different control reactions yet I cant figure it out. Anyone has ever had this problem? Any suggestions? what do you think? pleae HELP! it has been more than 5 weeks now and it is delaying all my work.
What strain of E. coli are you isolating the DNA from? endA+ strains will co-purify endonuclease with the plasmid DNA which will degrade the plasmid under restriction enzyme cutting conditions. You can reduce this problem by using the extra endonuclease wash steps in the Qiagen protocol, or (better) use a cloning strain which is endA-.
You could also be overloading your gels, which sometimes looks like degradation. I don't know what you are attempting to do with the boiling, but I would avoid it.
You could also be overloading your gels, which sometimes looks like degradation. I don't know what you are attempting to do with the boiling, but I would avoid it.
I am using OneSHot which are endA- yet I used the QBC buffer to clean up the DNA jsut in case in some runs.
I boiled the plasmid to try to get rid of any "nucleases" before cutting... it was just something that was worth trying.
Any further suggestions?
How long was this boil? Is the DNA still okay after that?
What solution do you use to elute DNA? Have you used TE or water. TE might be better. You could autoclave the solution or water to be extra cautious.
also try the extra wash step in the qiagen kit or try different cells.
I hope somebody can help me with what I've been going thruogh for the last 5 weeks. I have been getting smears of DNA after digesting my plasmid with different enzymes. I even dont have to put an enzyme and jsut by adding a bfufer I get smears. I did this for 2 hours and O/N incubation and still the same thing. I tried using a plasmid that I knew it worked ebfore and I got smears again for reasons I dont know. I boiled the plasmid to make sure to get rid of DNA it didnt help. I changed all water, buffer, tubes, tips, bench utensils and still I am ahving this problem. I re-transformed and re-purified the DNA (using QIAGEN miniprep kit) yet I am still havingt the same problem. The uncut looks perfect when I just add water (no buffer or BSA) however as soon as I add buffer and incubate it, I get smears. It is jsut crazy. It is something truly unbelievable. I have literally ran more tahn 200 different control reactions yet I cant figure it out. Anyone has ever had this problem? Any suggestions? what do you think? pleae HELP! it has been more than 5 weeks now and it is delaying all my work.
Is it just this particular DNA or other DNA that you've tried? Is anyone else in your lab having this problem? You need to isolate the problem, whether its general DNase contamination that is all over in the lab, just in your stuff, in your running buffers, or is it particular to this plasmid (in this case, the suggestions above apply). If it is all over in the lab, try to move to another lab to do your experiment (with their equipment). If you think it is just in your stuff try to use someone else's pippettors and/or use barrier tips. I know that they sell Rnase away. I'm not sure if they sell DNase away too.
I hope somebody can help me with what I've been going thruogh for the last 5 weeks. I have been getting smears of DNA after digesting my plasmid with different enzymes. I even dont have to put an enzyme and jsut by adding a bfufer I get smears. I did this for 2 hours and O/N incubation and still the same thing. I tried using a plasmid that I knew it worked ebfore and I got smears again for reasons I dont know. I boiled the plasmid to make sure to get rid of DNA it didnt help. I changed all water, buffer, tubes, tips, bench utensils and still I am ahving this problem. I re-transformed and re-purified the DNA (using QIAGEN miniprep kit) yet I am still havingt the same problem. The uncut looks perfect when I just add water (no buffer or BSA) however as soon as I add buffer and incubate it, I get smears. It is jsut crazy. It is something truly unbelievable. I have literally ran more tahn 200 different control reactions yet I cant figure it out. Anyone has ever had this problem? Any suggestions? what do you think? pleae HELP! it has been more than 5 weeks now and it is delaying all my work.
Is it just this particular DNA or other DNA that you've tried? Is anyone else in your lab having this problem? You need to isolate the problem, whether its general DNase contamination that is all over in the lab, just in your stuff, in your running buffers, or is it particular to this plasmid (in this case, the suggestions above apply). If it is all over in the lab, try to move to another lab to do your experiment (with their equipment). If you think it is just in your stuff try to use someone else's pippettors and/or use barrier tips. I know that they sell Rnase away. I'm not sure if they sell DNase away too.
Actually my other lab member just repeated the same hting using her own stuff and it worked excellent with her. I will try changing the pipets since all my buffers and everything else was just new.... i was the first to use.
Thank you all and will let you know for further problem occurs with that. I hope changing the pipets will be the solution